The P450 epoxygenase pathway metabolizes arachidonic acid (AA) into eicosanoid metabolites referred to as epoxyeicosatrienoic acids (EETs). EETs are produced in brain and perform important functions, including protection of neurons from ischemic injury. The major regio-isomer of EETs produced in brain and astrocytes is 14,15-EET. However, 14,15-EET is a preferred substrate for the soluble epoxide hydrolase (sEH), which metabolizes EETs into less active hydration products called dihydroxyeicosatrienoic acids (DHETs). Therefore, in the current proposal, we will test the hypothesis that sEH inhibition, effectively prolonging the half-life of 14,15-EET, is protective against ischemic brain injury in vivo. We will use selective inhibitors of sEH and sEH knockout (sEHKO) mice to determine if sEH inactivation reduces tissue damage and improves functional recovery after MCA occlusion (MCAO). We will combine EETs measurements in brain with pharmacological inhibitors of EETs synthesis and action in order to link protection acquired by sEH inactivation specifically to 14,15-EET. Furthermore, we will determine the cellular localization and effects of ischemia on the level and regional and subcellular distribution of sEH in ischemic brain. Finally, we will determine if neuroprotection by 14,15-EET is mediated in part via its action on putative membrane- associated receptor to specifically activate G-protein G alpha-s, leading to increased phosphorylation of cAMP-response element binding protein (CREB) and enhanced CRE-dependent gene transcription. The proposed studies are novel in that the pathophysiological role of sEH in cerebral ischemia has not been previously examined. The studies are also clinically relevant, and represent the first evaluation of a new class of sEH inhibitors as neuroprotective agents against brain damage after stroke.